Two-phase alocohol dehydrogenase-based coupled enzymatic reaction system

ABSTRACT

The present process relates to a coupled enzymatic reaction system which is implemented in a two-phase system consisting of organic phase and aqueous phase. The system operates with cofactor-dependent enzymes, the cofactor being continually regenerated enzymatically. The key enzyme is the alcohol dehydrogenase.

The present invention relates to a coupled reaction system operatingenzymatically which is distinguished in that it is implemented in asolvent mixture having two phases. In particular, the invention isdirected towards a reaction system comprising a cofactor-dependentenzymatic transformation of an organic compound and an enzymaticcofactor regeneration in the same system.

The isolation of optically active organic compounds, for examplealcohols and amino acids, by biocatalytic means is gaining increasinglyin importance. The coupled use of two dehydrogenases with cofactorregeneration has proved to be a method for the large-scale industrialsynthesis of these compounds (DE 197 53 350).

In situ regeneration of NADH with the NAD-dependent formatedehydrogenase in the course of the reductive amination of trimethylpyruvate to L-pseudoleucine (Bommarius et al. Tetrahedron Asymmetry1995, 6, 2851-2888).

Besides their catalytic property and efficiency, the biocatalysts thatare employed efficiently in the aqueous medium additionally have theadvantage, in contrast with a large number of synthetic metalliferouscatalysts, that the use of metalliferous feed materials, in particularfeed materials that contain heavy metal and are consequently toxic, canbe dispensed with. The use of expensive and, in addition, hazardousreducing agents such as borane, for example, in the course of theasymmetric reduction can also be dispensed with.

However, difficulties arise in the course of the conversion ofsubstrates that are sparingly soluble in water. Similar difficultiesexist in connection with sparingly water-soluble products. This is thecase, in particular, in the preparation of optically active alcohols inaccordance with the above concept, since the ketones that are requiredas starting compounds have a clearly lower solubility than the α-ketoacids employed in Scheme 1.

One conceivable solution in principle would be the implementation of thebiocatalytic reduction in a polar organic solvent or in an aqueoussolution thereof. In this case, both the enzymes and the substrate andoptionally the product should be soluble. A general disadvantage of adirect presence of an organic solvent, however, is constituted by theconsiderable diminution of the enzyme activity which generally occursunder these conditions (see, for example, Anderson et al., Biotechnol.Bioeng. 1998, 57, 79-86). Precisely formate dehydrogenase and, inparticular, the FDH derived from Candida boidinii or a mutant derivedtherefrom, being the only NADH regeneration enzyme employed hitherto onan industrial scale and available in commercial quantities, regrettablyhas high sensitivity to organic solvents (EP 1 211 316). This is alsoshown in Comparative Examples 1 to 8 with the use of DMSO, sulfolane,MTBE, acetone, isopropanol and ethanol etc. by way of organic solventcomponent in supplemental amounts of 10% in each case (see FIG. 1).

Various approaches are known with a view to solving this problemrelating to a stabilisation of the formate dehydrogenase derived fromCandida boidinii in the presence of organic solvents, for example theimplementation of reactions through additional use of tensides by way ofsurface-active substances. But the rate of reaction that is diminishedby a factor of approximately 40 (!) and also the inhibition of theformate dehydrogenase which occurs (B. Orlich et al., Biotechnol.Bioeng. 1999, 65, 357-362.) prove to be disadvantageous in this case.The authors note in addition that, by reason of the low stability of thealcohol dehydrogenase, a reduction process under these conditions of amicroemulsion is not economic. The same also holds, in principle, forthe method presented in EP 340 744, in which lyotropic mesophases werechosen as reaction site in the presence of an aqueous and/or organicphase.

A further basic possibility for the implementation of biocatalyticreactions consists in the application of immobilised enzymes in theorganic solvent or the use of enzymes in a homogeneous solutionconsisting of water and a water-miscible organic solvent. However, thesuccesses with these techniques, in which a direct contact of organicsolvent and enzyme occurs, are limited to a few enzyme classes, inparticular hydrolases. For instance, in DE 44 36 149 it is noted thatthe “direct presence of organic solvents (water-miscible ornon-water-miscible) is tolerated only by a few enzymes that pertain tothe class of the hydrolases.” Although a few further examples from otherenzyme classes have become known in the meantime (inter alia,oxynitrilases and an FDH derived from yeast), the statement made in DE44 36 149 continues to have validity for the majority of enzymes.Furthermore, an efficient immobilisation of the FDH derived from Candidaboidinii is not known. In addition, the immobilisation itself isassociated with additional costs due to the immobilisation step as wellas the immobilisation materials.

Therefore processes have been developed industrially that avoid thepresence of organic solvents by reason of the risk of the deactivationor denaturation of the enzymes. For instance, DE 44 36 149 describes aprocess in which the product is extracted from the reaction solutioninto an organic solvent through a product-permeable membrane, inparticular a hydrophobic membrane. Compared with a standard process in astirred-tank reactor, however, this process is technically clearly moreelaborate; besides, the requisite organic membranes are also anadditional cost factor. Furthermore, this method is only suitable forcontinuous processes. Moreover, it is a disadvantage that the achievablespace-time yields with this procedure are comparatively low. Forexample, in the course of the reduction of acetophenone a space-timeyield of only 88 g/(L*d) is obtained (S. Rissom et al., Tetrahedron:Asymmetry 1999, 10, 923-928). In this regard it is to be noted thatacetophenone itself is a relatively well water-soluble ketone, and mostanalogue substituted acetophenone ketones and related ketones possessfar lower solubilities, so that the space-time yields for typicalhydrophobic ketones should be distinctly lower. Despite theseconsiderable disadvantages, this process is regarded as the hithertopreferred method for the asymmetric biocatalytic reduction of sparinglysoluble ketones using isolated enzymes (see also: A. Liese, K. Seelbach,C. Wandrey, Industrial Biotransformations, Wiley-VCH Verlag, Weinheim,2000, pp. 103-106).

In the doctoral thesis by Tien Van Nguyen (Rheinisch-WestfälischeTechnische Hochschule Aachen, 1998), inter alia a reaction system isdescribed consisting of alcohol dehydrogenases, NADH, formatedehydrogenase in the heptane/water solvent system for the reduction ofp-chloroacetophenone. Here the substrate was employed in each case inconcentrations of 10 mmol per L of total volume of the solvents (=sum ofthe volumes of organic solvent and aqueous portion). According to theresults, only up to these substrate concentrations is it possible foracceptable yields of product to be obtained. Such a substrateconcentration of 10 mM or below is, however, nowhere near sufficient foran industrial application. The space-time yields resulting therefromwould be much too low for an industrial application.

This assessment by T. N. Nguyen with respect to the problems at highersubstrate concentrations is, incidentally, confirmed also in numerousother reference publications, which has led to the aforementionedattempted solutions, for example by using membranes.

In addition, within the scope of their paper on the biocatalyticpreparation of a pharmaceutical active substance by reduction of aketone, Anderson et al. point to a (generally to be expected) furtherdisadvantage at higher substrate concentrations, namely toxicity effectswhich occur widely precisely in the case of the hydrophobic alcohols. Inthis paper (B. A. Anderson et al., J. Am. Chem. Soc. 1995, 117,12358-12359) it is stated that—in contrast with activity tests—reactionson a preparative scale, that is to say with “acceptable substrateconcentrations”, prove to be problematic as a result of considerabletoxicity effects. In this case these effects were noted even withenzymes “immobilised” in cells and, in addition, were observed inaqueous solution. As expected, corresponding inhibitions at a highersubstrate concentration should accordingly occur to an increased extentin the case where use is made of “free” isolated enzymes and in thepresence of organic solvents.

To sum up, it may consequently be noted that at the moment no process isknown that helps to circumvent the disadvantages listed above andpermits the enzymatic preparation of sparingly water-soluble substrateson an industrial scale.

The object of the present invention was therefore to specify apossibility as to how, in particular, sparingly water-soluble organiccompounds can be made available to a coupled cofactor-dependentenzymatic conversion to such a sufficient extent that an application ofthe conversion on an industrial scale can be undertaken undereconomically and ecologically advantageous conditions. In particular,one object was that such a process should be suitable for the reductionof sparingly water-soluble ketones.

This object is achieved in the manner defined in the Claims. Claims 1 to10 are directed towards a reaction system operating in accordance withthe invention. Claim 11 protects a device. Claim 12 relates to a processoperating in accordance with the invention, whereas claims 13 and 14 aredirected towards preferred uses of the reaction system according to theinvention.

By virtue of the fact that a coupled enzymatic reaction system is madeavailable having a cofactor-dependent enzymatic transformation of anorganic compound with an alcohol dehydrogenase and an enzymaticregeneration of the cofactor in a two-phase solvent system in which anaqueous phase is in contact with a liquid organic phase and the organiccompound is present in a concentration of >25 mM per L of total volumeof the solvents (=sum of the volumes of organic solvent and aqueousportion), the solution to the stated object is attained, in particularin a manner that is surprising, by no means foreseeable and, accordingto the invention, particularly advantageous. Contrary to the opinionthat can be deduced from the prior art, it is surprisingly possible,despite the presence of an organic solvent, to allow the coupledenzymatic reaction system to operate without loss of activity, due tothe solvent, of one of the enzymes in concentrations that are sufficientfor the industrial scale.

The organic solvent that is employed in the reaction system is intendedto form two separate phases with the aqueous phase that is present.Within the bounds of this requirement, a person skilled in the art is,in principle, free in the choice of the organic solvent. However, it hasproved to be advantageous if by way of organic phase a solvent is chosenthat possesses a solubility in water that is as low as possible (logPvalue ≧3, preferably ≧3.1, more preferably ≧3.2 etc.). Since the organicsolvent is also intended to take up the sparingly water-soluble educt atthe same time, it is also important furthermore that said solventpossesses a solubility in respect of the organic compounds employed thatis as high as possible. Organic solvents of such a type, which can bepreferably employed in the reaction system, are aromatic or aliphatichydrocarbons that are liquid under the given reaction conditions. Inparticular, toluene, xylenes, benzene, n-pentane, n-hexane, n-heptane,n-octane, isooctane, cyclohexane, methylcyclohexane and alsobranched-chain isomers thereof are most particularly preferred.Halogenated hydrocarbons can also be employed (CHCl₃, CH₂Cl₂,chlorobenzene etc.).

The quantitative ratio of organic solvent to aqueous portion can bechosen arbitrarily. The organic solvent is employed in a quantityrelative to the total volume of the solvents (=sum of the volumes oforganic solvent and aqueous portion) amounting to 5-80 vol. %,preferably 10-60 vol. %, particularly preferably 20-50 vol. %.

Contrary to the approach that is proposed in the prior art, namely ofadding surfactants to the enzymatic reaction mixture in order toaccelerate the enzymatic transformation, in which phase transitions inthe course of the reaction are minimised, the present invention providesevidence that the use of a reaction system according to the inventionproceeds particularly successfully when the system contains nosurfactants.

The term ‘surfactants’ in this context is understood to mean all thosesubstances which are capable of building up micellar structures or oflowering the surface tension at liquid-liquid phase boundaries.

As already indicated, the concentration with which the substrates areemployed in the reaction system should be such that a conversion can beeffected that is advantageous from economic viewpoints. The organiccompound should therefore be present prior to the start of the reactionadvantageously in a concentration of >25 mM, preferably >100 mM,particularly preferably >200 mM and most particularly preferably >500 mMper L of total volume of the solvents (=sum of the volumes of organicsolvent and aqueous portion). An upper limit for the concentration isconstituted naturally by the guarantee of the viability of the reaction;in particular, stirrability of the reaction mixture should obtain inevery case. However, working may preferably take place also above thesaturation limit for the substrate or the product.

Cofactors are familiar to a person skilled in the art (Enzyme Catalysisin Organic Synthesis, Ed.: K. Drauz, H. Waldmann, 1995, Vol I, p. 14,VCH). For the redox reactions to be catalysed, the alcoholdehydrogenases to be considered here preferably utilise, by way ofcofactors, molecules such as, for example, NAD, NADH, NADPH or NADP ashydrogen-carriers.

The stated coupled enzymatic reaction system can, according to theinvention, be employed in all enzymatic reactions coming intoconsideration by a person skilled in the art for this purpose in whichketo groups are converted into alcohol groups. Preferred, however, areoxidoreductase reactions, as stated. The alcohol dehydrogenases that areemployed in accordance with the invention preferably originate fromthe-organisms Rhodococcus erythropolis (S-ADH) or Lactobacillus kefir(R-ADH) (Nguyen Doctoral Thesis, Aachen, 1998).

The enzyme that regenerates the cofactor employed is, in principle,dependent on the cofactor employed, but on the other hand also on thecosubstrate to be oxidised or reduced. In Enzyme Catalysis in OrganicSynthesis, Ed.: K. Drauz, H. Waldmann, 1995, Vol I, VCH, p. 721 a numberof enzymes for the regeneration of NAD(P) are named. For these reasonsso-called formate dehydrogenase (FDH, Scheme 1), which is of interestcommercially and also obtainable on a large scale as well as beingemployed at present for the synthesis of amino acids, is advantageouslyemployed. It should therefore be used preferentially for theregeneration of the cofactor. In most particularly preferred manner theFDH originates from the organism Candida boidinii. Further-developedmutants of the same can also be employed (DE 197 53 350). Particularlysurprising in this case is the fact that the formate dehydrogenasederived from C. boidinii can be employed efficiently under theseconditions despite the high instability in relation to organic solvents(see Comparative Examples in the Experimental Part) that is observed.

A so-called NADH oxidase derived from, for example, Lactobacillus kefiror Lactobacillus brevis can likewise be employed for the regeneration ofNADH.

In a next development the present invention relates to a device for thetransformation of organic compounds that has the reaction systemaccording to the invention. Devices to be employed advantageously are,for example, the stirred tank or stirred-tank cascades, or membranereactors that can be operated both in batch operation and continuously.Within the scope of the invention the term ‘membrane reactor’ isunderstood to mean any reaction vessel in which the catalyst is enclosedin a reactor while low-molecular substances are supplied to the reactoror are able to leave it. In this connection the membrane may beintegrated directly into the reaction chamber or may be installedoutside in a separate filtration module wherein the reaction solutionflows continuously or intermittently through the filtration module andthe retentate is recycled into the reactor. Suitable embodiments aredescribed, inter alia, in WO 98/22415 and in Wandrey et al. in Jahrbuch1998, Verfahrenstechnik and Chemieingenieurwesen, VDI p 151 ff.; Wandreyet al. in Applied Homogeneous Catalysis with Organometallic Compounds,Vol. 2, VCH 1996, p 832 ff.; Kragl et al., Angew. Chem. 1996, 6, 684 f.The continuous mode of operation which is possible in this apparatus inaddition to the batch and semicontinuous modes of operation can beimplemented as desired in the cross-flow filtration mode (FIG. 3) or inthe form of dead-end filtration (FIG. 2). Both process variants aredescribed in principle in the prior art (Engineering Processes forBioseparations, Ed.: L. R. Weatherley, Heinemann, 1994, 135-165; Wandreyet al., Tetrahedron Asymmetry 1999, 10, 923-928).

A next development of the invention is concerned with a process for theenzymatic transformation of organic compounds by application of thereaction system according to the invention. The process is preferablyone involving the preparation of an enantiomer-enriched organiccompound, preferably a chiral alcohol. The design of the process can beworked out at the discretion of a person skilled in the art on the basisof the reaction system that has been described and the examples that arepresented below. Under the given boundary conditions, the conditionsthat are otherwise known for the enzymatic conversion are setappropriately.

A next aspect of the invention is concerned also with the use of thereaction system according to the invention in a process for theenzymatic transformation of organic compounds or for the diagnosis oranalysis of organic compounds, preferably of alcohols. In furtherpreferred manner the reaction system according to the invention is, asstated, employed in a process for the preparation of enantiomer-enrichedorganic compounds, preferably of alcohols.

The expression ‘coupled enzymatic system’ is understood to mean,according to the invention, that an enzymatic transformation of anorganic compound takes place subject to consumption of a cofactor andthe cofactor is regenerated in situ by a second enzymatic system. As aresult, this leads to a diminution of the use of expensive cofactors.

The present invention can be elucidated on the basis of the exampleprovided by the alcohol-dehydrogenase/ NADH/FDH/formic-acid system. Theasymmetric synthesis of alcohols was carried out by means of thisreaction system, starting from the corresponding ketone.

Processing of the reaction mixture was effected by extraction with MtBEand concentration of the organic phase by evaporation. The correspondingalcohol was obtained in this way in a very simple manner in terms ofapparatus with a conversion of 69% and with an enantioselectivity of 99%(Example 3).

But outstanding enantioselectivities are also obtained with the use ofother ketones as starting materials. For instance, the reduction ofphenoxyacetone under these reaction conditions results in an enantiopureproduct quantitatively with >99.8% ee (Example 4).

But the reaction system according to the invention is also suitable,moreover, for sterically demanding ketones. This will be documented inexemplary manner on the basis of the example provided by α,m-dichloroacetophenone. This ketone is substituted by a chlorine atomboth on the methyl group and on the aromatic ring. The biocatalyticreduction in the 2-phase system here yields the desired product2-chloro-1-(m-chlorophenyl)ethanol, again with outstandingenantioselectivity of >99.2% (Example 5). The conversion here is around77%.

The corresponding experiments of experimental Examples 3-5 are presentedin Scheme 2.

These high conversions and enantioselectivities are surprising, notleast for the reason that, by virtue of the presence of organicsolvents, often not only a diminution of the enzyme activity(accompanied by a low conversion) but also a change in enzyme propertieswith regard to stereospecificity (accompanied by a diminution ofenantioselectivity) is to be observed.

In this context, however, the results of the experiments at elevatedsubstrate concentrations turned out to be particularly surprising. Theseexperiments were carried out with p-chloroacetophenone as modelsubstrate. If in the above experiment at a substrate concentration of 10mM (this substrate concentration corresponds to the concentration in thecase of the experiments from the prior art) a conversion of 69% isachieved (Example 3), then this conversion—contrary to the widespreadview that at elevated substrate concentrations only diminished yieldscan be achieved, by reason of inhibitions etc.—was able to be increasedwith this type of reaction, starting now from a concentration relativeto the total volume of solvents (=organic and aqueous solvents) of >25mM, and higher conversions of 75% (at 40 mM) and 74% (at 100 mM) couldbe achieved (Examples 6, 7).

In this connection the high conversion at a concentration of 100 mM(Example 7) is particularly worth mentioning.

The experiments relating to enzymatic reduction at varying substrateconcentrations (Examples 3, 6, 7) are presented graphically in Scheme 3and FIG. 4.

In further experiments the long-term stability of the FDH derived fromC. boidinii in various solvent systems was investigated. In contrastwith most organic solvents (see Comparative Examples), which lead to arapid deactivation of the FDH, in the two-phase system, particularlywhen use is made of the aforementioned hydrocarbon components,outstanding stability properties of the formate dehydrogenase, inparticular of the FDH derived from C. boidinii, were still observed evenafter several days. Whereas, for example, in the presence of acetone orDMSO the enzyme activity declines within 24 hours by 35% or 66%,respectively, in the presence of 20 vol. % hexane 90% enzyme activitycan still be registered even after 3 days. The results with n-hexane arereproduced in FIG. 1, represented graphically, and in Table 3. TheComparative Examples with other organic solvents are likewise recordedin FIG. 1.

A principal advantage of this process consists in its simplicity. Forinstance, no elaborate process steps are included, and the process canbe implemented both in batch reactors and continuously. Similarly, incontrast with earlier processes, no special membranes which separate theaqueous medium from the organic medium are required. The additions ofsurfactant which are required in some previous processes also becomeunnecessary with this process. A further principal advantage consists inthe first-time possibility of organising the enzymatic preparation ofoptically active alcohols in technically meaningful substrateconcentrations of >25 mM. These advantages could not be deduced inobvious manner from the prior art.

The term ‘enantiomer-enriched’ designates the fact that one opticalantipode is present in the mixture with its other one in a proportionamounting to >50%.

In the case where one stereocentre is present, the structures that arerepresented relate to both of the possible enantiomers and, in the casewhere more than one stereocentre is present in the molecule, to allpossible diastereomers and, with respect to one diastereomer, to thepossible two enantiomers of the compound in question which areencompassed thereby.

The organism C. boidinii is deposited in the American Type CultureCollection under number ATCC 32195 and is publicly accessible.

The documents of the prior art that have been named in this publicationare considered as being jointly encompassed by the disclosure.

DESCRIPTIONS OF THE DRAWINGS

FIG. 1 shows a membrane reactor with dead-end filtration. The substrate1 is transferred into the reactor chamber 3, which has a membrane 5, viaa pump 2. Located in the stirrer-driven reactor chamber, in addition tothe solvent, are the catalyst 4, the product 6 and unconverted substrate1. Principally low-molecular product 6 is filtered off via the membrane5.

FIG. 2 shows a membrane reactor with cross-flow filtration. Thesubstrate 7 here is transferred via the pump 8 into the stirred reactorchamber in which solvent, catalyst 9 and product 14 are also located.Via the pump 16 a flow of solvent is set which, via an optionallypresent heat-exchanger 12, leads into the cross-flow filtration cell 15.Here the low-molecular product 14 is separated via the membrane 13.High-molecular catalyst 9 is subsequently conducted back into thereactor 10 with the flow of solvent, optionally again via aheat-exchanger 12, optionally via the valve 11.

EXPERIMENTAL PART Example 1 Comparative Examples of the FDH ActivitiesUsing an FDH Derived from C. boidinii (Double Mutant: C23S/C262A)

2.72 g (0.8 mol/L) sodium formate and 1.14 g (0.1 mol/L) dipotassiumhydrogenphosphate trihydrate are weighed out and dissolved in 40 mLfully demineralised H₂O . With ammonia solution (25%) and formic acid(100%), or corresponding diluted solutions, the pH value of the solutionis set to 8.2. Then the solution is transferred into a 50 mL measuringflask and topped up with fully demineralised H₂O. Separately from this,71.7 mg (4 mmol/L) NAD⁺ trihydrate are weighed out and dissolved inabout 20 mL fully demineralised H₂O. With ammonia solution (25%) andformic acid (100%), or corresponding diluted solutions, the pH value ofthe solution is set to 8.2. Then the solution is transferred into a 25mL measuring flask and topped up with fully demineralised H₂O.Subsequently, in each case, 500 μL of the substrate solution and also ofthe NADH solution are mixed in the 1 cm cell which is used for themeasurement. After addition of 10 μL of the enzyme solution, whereby a10% solution of an organic solvent (see Table) in water findsapplication by way of solvent, shaking is effected briefly, the cell isplaced into the photometer, and the recording of data is started. Theenzyme solution is firstly added directly prior to the start ofmeasurement. The activities of the FDH derived from C. boidinii (doublemutant: C23S/C262A) are determined after certain periods of time by thephotometric detection of the reaction of NAD⁺ to form NADH. Thephotometric measurement was undertaken at a temperature of 30° C., at awavelength of 340 nm and with a measuring-time of 15 min. The resultsare represented below in Table 1 and Table 2. TABLE 1 Enzyme activity ofthe FDH derived from C. boidinii (double mutant: C23S/C262A) in U/mL asa function of solvent and time Aceto- Butanol MEK DMSO THF Sulfolanenitrile Time Activity Activity Activity Activity Activity Activity [d][U/ml] [U/ml] [U/ml] [U/ml] [U/ml] [U/ml] 0.000 0.5262 0.0058 0.79650.8492 0.0028 0.7961 0.042 0.0006 0.0011 0.7880 0.4357 0.0003 0.44940.125 0.7794 0.0414 0.0840 1.097 0.2669 0.0008 2.035 0.2331 2.896 0.22015.927 0.1763 7.885 0.1404 9.948 0.1205 13.073 0.0915 14.892 0.071716.875 0.0540 19.938 0.0355

TABLE 2 Enzyme activity of the FDH derived from C. boidinii (doublemutant: C23S/C262A) in U/mL as a function of solvent and time TimeAcetone Ethanol [d] Activity [U/ml] Activity [U/ml] 0.000 0.8355 0.84910.042 0.7402 0.7689 0.750 0.5893 0.6367 1.000 0.5426 0.5933 1.875 0.34840.4687 2.760 0.2691 0.3510 3.781 0.2004 0.2814 4.646 0.1614 0.2240 5.8750.1325 0.1736 6.778 0.0987 0.1486 7.792 0.0794 0.1277 8.729 0.06100.0998 11.750 0.0333 0.0536 13.726 0.0421

Example 2 Measurement of the FDH Activities

Determination of the activity was undertaken in accordance with theinstructions in Example 1, with hexane being used as organic solventcomponent. The results are represented below in Table 3. TABLE 3 Enzymeactivity of the FDH derived from C. boidinii (double mutant: C23S/C262A)in U/mL as a function of hexane and time Time Hexane (10%) Hexane (20%)[d] Activity [U/ml] Activity [U/ml] 0.000 0.8364 1.0280 0.042 0.95720.9952 0.177 0.8223 1.1408 0.899 0.7892 0.9311 2.000 0.6242 0.9467 2.8780.7654 0.9280

Example 3 Conversion with p-chloroacetophenone

To a solution consisting of p-chloroacetophenone (78.4 mg; 10 mM),sodium formate (50 mM) and NADH (2 mM) in 10 mL n-heptane and 40 mL of aphosphate buffer, 10.1 U of alcohol dehydrogenase (derived fromRhodococcus erythropolis) and 10 U of a formate dehydrogenase (FDHderived from C. boidinii, expression in E. coli, double mutantC23S/C262A) are added. The reaction mixture that has arisen is left tostir for 21 hours at 30° C. Subsequently processing proceeds viaextraction with 3×25 mL MTBE, and the collected organic phases are driedwith sodium sulfate. The crude product resulting after removal of thesolvent in a vacuum is examined with regard to conversion (by ¹H-NMRspectroscopic examination) and enantioselectivity (by chiral GC).

Conversion: 69%

Enantioselectivity: >99% ee

Example 4 Conversion with phenoxyacetone

To a solution consisting of phenoxyacetone (76.0 mg; 10 mM), sodiumformate (50 mM) and NADH (2 mM) in 10 mL n-heptane and 40 mL of aphosphate buffer, 10.1 U of alcohol dehydrogenase (derived fromRhodococcus erythropolis) and 10 U of a formate dehydrogenase (FDHderived from C. boidinii, expression in E. coli, double mutantC23S/C262A) are added. The reaction mixture that has arisen is left tostir for 21 hours at 30° C. Subsequently processing proceeds viaextraction with 3×25 mL MTBE, and the collected organic phases are driedwith sodium sulfate. The crude product resulting after removal of thesolvent in a vacuum is examined with regard to conversion (by ¹H-NMRspectroscopic examination) and enantioselectivity (by. chiral GC).

Conversion: >95%

Enantioselectivity: >99.8% ee

Example 5 Conversion with 2,3′-dichloroacetophenone

To a solution consisting of 2,3′-dichloroacetophenone (102.7 mg; 10 mM),sodium formate (50 mM) and NADH (2 mM) in 10 mL n-heptane and 40 mL of aphosphate buffer, 10.1 U of alcohol dehydrogenase (derived fromRhodococcus erythropolis) and 10 U of a formate dehydrogenase (FDHderived from C. boidinii, expression in E. coli, double mutantC23S/C262A) are added. The reaction mixture that has arisen is left tostir for 21 hours at 30° C. Subsequently processing proceeds viaextraction with 3×25 mL MTBE, and the collected organic phases are driedwith sodium sulfate. The crude product resulting after removal of thesolvent in a vacuum is examined with regard to conversion (by ¹H-NMRspectroscopic examination) and enantioselectivity (by chiral GC).

Conversion: 77%

Enantioselectivity: >99.2% ee

Example 6 Conversion with p-chloroacetophenone at 40 mM

To a solution consisting of p-chloroacetophenone (78.4 mg; 10 mM),sodium formate (50 mM) and NADH (2 mM) in 2.5 mL n-heptane and 10 mL ofa phosphate buffer, 10.1 U of alcohol dehydrogenase (derived fromRhodococcus erythropolis) and 10 U of a formate dehydrogenase (FDHderived from C. boidinii, expression in E. coli, double mutantC23S/C262A) are added. The reaction mixture that has arisen is left tostir for 21 hours at 30° C. Subsequently processing proceeds viaextraction with 3×25 mL MTBE, and the collected organic phases are driedwith sodium sulfate. The crude product resulting after removal of thesolvent in a vacuum is examined with regard to conversion (by ¹H-NMRspectroscopic examination) and enantioselectivity (by chiral GC).

Conversion: 75%

Example 7 Conversion with p-chloroacetophenone at 100 mM

To a solution consisting of p-chloroacetophenone (78.4 mg; 10 mM),sodium formate (50 mM) and NADH (2 mM) in 1 mL n-heptane and 4 mL of aphosphate buffer, 10.1 U of alcohol dehydrogenase (derived fromRhodococcus erythropolis) and 10 U of a formate dehydrogenase (FDHderived from C. boidinii, expression in E. coli, double mutantC23S/C262A) are added. The reaction mixture that has arisen is left tostir for 21 hours at 30° C. Subsequently processing proceeds viaextraction with 3×25 mL MTBE, and the collected organic phases are driedwith sodium sulfate.

The crude product resulting after removal of the solvent in a vacuum isexamined with regard to conversion (by ¹H-NMR spectroscopic examination)and enantioselectivity (by chiral GC).

Conversion: 74%

1. A coupled enzymatic reaction system having a cofactor-dependentenzymatic transformation of an organic compound with an alcoholdehydrogenase and an enzymatic regeneration of the cofactor in atwo-phase solvent system, wherein an aqueous phase is in contact with aliquid organic phase and the organic compound is present in aconcentration of >25 mM per L of total volume of the solvents. 2.Reaction system according to claim 1, characterised in that the organicsolvent employed possesses a solubility in water that is as low aspossible and a solubility in respect of the organic compounds employedthat is as high as possible.
 3. Reaction system according to claim 1and/or 2, characterised in that aromatic or aliphatic hydrocarbons thatare liquid under the reaction conditions are employed as organicsolvent.
 4. Reaction system according to one or more of the precedingclaims, characterised in that the organic solvent is present in aquantity amounting to 5-80 vol. % in relation to the total volume of thesolvents.
 5. Reaction system according to one or more of the precedingclaims, characterised in that the system contains no surfactants. 6.Reaction system according to one or more of the preceding claims,characterised in that the organic compound is present prior to the startof the reaction in a concentration of >100 mM per L of total volume ofthe solvents.
 7. Reaction system according to one or more of thepreceding claims, characterised in that NADH or NADPH is employed ascofactor.
 8. Reaction system according to one or more of the precedingclaims, characterised in that an alcohol dehydrogenase derived fromLactobacillus kefir is employed as enzyme for the transformation of theorganic compound.
 9. Reaction system according to one or more of claims1 to 7, characterised in that an alcohol dehydrogenase derived fromRhodococcus erythropolis is employed as enzyme for the transformation ofthe organic compound.
 10. Reaction system according to one or more ofthe preceding claims, characterised in that regeneration of the cofactoris effected by a formate dehydrogenase, preferably that derived fromCandida boidinii or mutants thereof.
 11. A device for the transformationof organic compounds, having a reaction system according to one ofclaims 1 to
 10. 12. A process for the enzymatic transformation oforganic compounds by application of the reaction system according to oneof claims 1 to
 10. 13. Use of the reaction system according to one ofclaims 1 to 10 for the enzymatic transformation of organic compounds orfor the diagnosis or analysis of, preferably, alcohols.
 14. Useaccording to claim 13 in a process for preparing enantiomer-enrichedorganic compounds, preferably alcohols.